Current Agriculture Research Journal Vol. 2(2), 83-88 (2014)

Stability of Based Biopesticide in Operational Conditions in

Papa Madiallacké Diedhiou1*, Kemo Badji2, Rokhaya Faye3 and Papa Ibra Samb3

1Faculté d’Agronomie, Université Gaston Berger ; Saint-Louis - Sénégal. 2Plant Health and Quarantine Officer, Direction de la Protection des Végétaux, Dakar- Sénégal 3Département de Biologie Végétale, Université Cheikh Anta Diop de Dakar, Sénégal.

http://dx.doi.org/10.12944/CARJ.2.2.03

(Received: November 17, 2014; Accepted: November 25, 2014)

Abstract

Grasshoppers and are important pests in the Sahel zone with a big economic and environmental impact. The move towards biological control methods represents an important option to mitigate environmental impact linked with the use of insecticides for their control. Metarhizium acridum, a pathogenic fungus that gave excellent test results in controlling , was further developed as biopesticide. The stability of the manufactured biopesticide in operational conditions was tested. In the present study, the germination of spores and mycelial growth of M. acridum were higher at 28 °C and 33 °C. Higher temperatures led to a significant decrease of these biological parameters. The sporulation of M. acridum is abundant at 28 °C and 33 °C but was completely inhibited at 37 °C. Furthermore, M. acridum based biopesticide showed a good infection rate for the dry spores formulation for 3 months of storage in all test sites. In the oil formulation in contrast, infectivity rate dropped quickly to a very low level after one month storage in the inland sites and remained good in Dakar. High temperatures and low moisture, away from the coast were speculated to be the reasons.

Key words: Metarhizium acridum, Biopesticide, Grasshoppers, Locusts, Oil formulation,Dry spore formulation.

INTRODUCTION in 2004, a total of 1,040 billion CFA was needed to mitigate environmental pollution (Locustox, 2009). Grasshoppers and locusts are major pests in most countries of the Sahel zone like Senegal. Gradually, environment friendly alternatives In invasion years, their control requires annually pesticides are being developed to control locusts. large amounts of synthetic pesticides sprayed over Biopesticides like those having pathogenic about 1,000,000 ha per year (Launois Luong et al., microorganisms as active agent are increasingly 1988). In locust invasion years, about 2,000,000 developed for operational use in several countries ha are treated with pesticides to achieve a control of the Sahel zone. over the large migrating locusts and grasshoppers populations (Anon., 1997). Besides negative In West Africa, particularly in Senegal, economic impact, should be added other impacts Metarhizium acridum, a locust pathogenic fungus on the environment and on health of humans as gave excellent test results in controlling grasshoppers. well as on . With respect to negative impact The fungal strain was further formulated to a on environment, the decontamination of 1 ton of biopesticide, found to be very effective against sand contaminated with pesticides is estimated at locusts and grasshoppers. Those belong to 26,000,000 CFA. With a total of 40,000 tons of soil the order , and consist among others, to be decontaminated in pesticide storages sites with Schistocerca gregaria, Oedaleus senegalensis, 84 Diedhiou et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014)

Hieroglyphus daganensis, Locusta migratoria, etc. Study sites The spores of Metarhizium acridum represent the The study of the viability of the spores of active ingredient of the bioinsecticide, intended for Metarhizium acridum in different formulations of locust control. However, the widespread use of this the biopesticide during storage was carried out in biopesticide in operational conditions in Senegal is the storage facilities of “Direction de la Protection facing a technical problem due to a lack of stability des Végétaux” (DPV), the National Plant Protection of the active ingredient during storage. In fact, the Organization. Those storage facilities are located biological effectiveness of the product in operational in different cities scattered over the whole country, conditions drops below the critical rate of 85% like Richard Toll, Louga, Gossas, Nioro du Rip and infectivity (Jaronski, 2000) in different environments. Dakar. They are designed for almost permanent Nonetheless, the feasibility of this control method, in storage of insecticides for locust control targeting particular during the hot rainy season, characterized O. senegalensis and S. gregaria. The mean with rapid buildup of locust populations, and a temperatures and relative humidity recorded in the worsening of roads and transportation systems, storages facilities during the experiment are listed requires the pesticides to be stored in close proximity in Table 1. to the agrosystems at risk and applied as needed. Storage test The present work therefore was intended to Samples the biopesticide in dry spores look at the influence of temperature on the survival formulation as well as oil formulation were placed of the active ingredient of a Metarhizium acridum in test tubes. For dry spores formulation, 1 g of based biopesticide in different formulations, with powder was sampled and 5ml of oil formulation. In special focus on the storage facilities of the National each storage facility, 5 tubes for oil formulation and Plant Protection Organization over the country. 5 other tubes for dry spores formulation were kept. The germination rate was recorded before storage MATERIALS AND METHODS and every 30 days, as described by Moore et al., (1996). For the germination test, 1 ml was sampled The fungal biocontrol agent from oil formulation containing tubes and 0.1 g from Metarhizium acridum strain IMI 330189 is dry formulation treatment. The samples were kept the active ingredient of the biopesticide. It is a fungus in small vials closed with parafilm and placed in a of the class ascomycete with Metacordyceps sp humid chamber for 24 hours (Moore et al., 1996). as teleomorph. Species of the genus Metarhizium Thereafter, 10 ml sterile water and a droplet of live naturally in the soil litter almost everywhere in Tween 80 were added prior to centrifugation for 5 the world. Metarhizium acridum strain IMI 330189 minutes at 2500 rotations / min. The solvent was was isolated from a parasitized Ornithacris cavroisi gently removed and the pellet, composed with found in (Kooyman, 2007). The biopesticide is spores is mixed with 10 ml sterile water and Tween formulated as a powder composed with dry spores or 80. This spore suspension is used for germination spores mixed with vegetable oil as oil formulation. test by plating a 20, 50 and 100 µL into petri dishes containing Sabouraud Dextrose Agar culture media.

Table. 1: Mean temperatures and moisture in the storage facilities of DPV during the test period

Site hosting pesticide temperatures (°C) Relative humidity (%) storage facility Maxima Minima Mean Maxima Minima Mean

Gossas 37.00 23.63 29.69 66.70 32.10 59.58 Dakar 34.01 23.63 30.22 70.70 34.80 60.64 Louga 33.17 23.24 28.87 30.4 27.4 26.14 Nioro 37.88 24.01 30.13 28.52 22.6 24.97 R. Toll 37.88 22.48 29.58 25.42 16.61 22.68 Diedhiou et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) 85

For the oil formulation, the sampled volume was spores extracted from the biopesticide. The number completed to 10 ml with sterile water and Tween 80. of germinated conidia was determined by over a 20 The mixture was centrifuged and the pellet collected conidia sample, repeated 10 times after 6 hours, 12 and suspended in 10 ml sterile water and Tween 80 hours, 18 hours and 24 hours of incubation. before being plated onto the culture media. Data analysis Effect of temperature on spore germination, An analysis of variance was performed mycelial growth and sporulation for data with a normal distribution. Data expressed For the study of mycelial growth and in percentages were first normalized by arcsinus sporulation ability, petri dishes with the culture media transformation. The statistical analysis software were inoculated with a 6 mm diameter of 4-5 days Minitab 13th edition for Windows was used. Means old culture of the fungus and incubated at different were separated using the Student test & Newman temperatures. For sporulation performance mature Keuls at 5 %. conidia were harvested after 3 weeks of incubation at constant temperatures in the dark. To collect the RESULTS spores, a fine brush and 10 ml sterile water and Tween 80 were used to prepare a suspension of Effect of different temperatures on germination spores. The concentration of the suspension of of spore of M. acridum spores obtained was determined using the grid cell The spore germination of M. acridum at of Neubauer (Tefera and Pringle, 2003). different temperatures gave variable results (Fig. 1). The highest rates were obtained at 33 °C and 28 °C Mycelial growth of the fungus at different with 18% and 21.12% respectively after only 6 hours temperatures was measured by taking the average of incubation. The germination rate reached over of two perpendicular diameters of colony in the petri 90% after 24h. When temperatures reached 35 °C dish. The diameter was measured every 24 hours and 37 °C, the germination rate did not cross the 10% for 11 days (Tefera and Pringle, 2003). bar after 24 hours. The analysis of variance showed a highly significant difference between treatments (p The effect of a range of constant H” 0.000). temperatures was evaluated on the germination of

Fig. 1: Influence of different temperatures on spore germination ofM. acridum (n = 10; p< 0.000) 86 Diedhiou et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014)

Effect of different temperatures on mycelial highly significant differences between treatments (p growth of M. acridum <0.001). A daily average mycelia growth of 3.648 mm was observed at 28 °C. It decreased down to Effect of temperature on the production of 1.93 mm at 33 °C, and further to 0.78 mm at 35 conidia °C and 0,012 °C at 37 mm. The colony diameter At 28 °C, about 3*108 spores per petri dish was subsequently the highest at 28 °C and 33 °C, were produced (Fig. 3). Spore production reached with respectively 40 mm and 25 mm after 12 days 6.8*107 spores per petri dish at 33 °C and was (Fig. 2). Colony size reached barely 10 mm and 6 suppressed at 35 °C and 37 °C. Variance analysis mm respectively at 35 °C and 37 °C after 12 days showed highly significant differences (p <0.001). of incubation. The analysis of variance showed

Fig. 2: Influence of different temperatures on the mycelial growth ofM. acridum (P <0.001)

Fig. 3: Production of spores by M. acridum under different incubation temperatures (n = 10; p < 0.001) Diedhiou et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) 87

Effect of storage in operational conditions on In the present study, germination of spores survival of M. acridum and mycelial growth of M. acridum are higher at 28 °C The storage test in DPV facilities over and 33 °C. Higher temperatures lead to a significant different sites in Senegal gave the results in figure 4. decrease of these biological parameters, with a The survival rate was higher for dry spores formulation survival of the biological agent compromised at 37 compared to oil formulation. This survival rate was °C. over 90% for spores in dry formulation in all sites. This level of survival rate of spores was recorded Locusts are known to hang at the top herbs for oil formulation only in Dakar. The general trend to enjoy the sun and raise their body temperature. for oil formulation shows a quick decrease from the If this behavior occurs at 37 °C, for a long period, second month of storage onwards to reach values the progression of fungal infections may slow down. below 30% at the end of the experiment, 90 days In fact, Ouedraogo (1996) and Dimbi et al., (2004) later. Variance analysis showed highly significant showed that mortality of migratory locust goes from differences between treatments (p <0.001). 78 % in the shade, down to 21% in the sun within 6 days. It should however be reminded that daily DISCUSSION temperatures in the Sahel are very variable and furthermore, locust are more frequent in habitats Fungal growth results from spore with milder temperatures. These environments are germination, mycelial growth and sporulation to more favorable for germination, mycelial growth and achieve a virulence and good dispersal over space sporulation of the fungus. and time. These parameters are often directly affected by climatic factors such as moisture level Sporulation of M. acridum is abundant at or temperature (Luz et al., 1998). 28 and 33 °C. It is completely inhibited at 37 °C,

Fig. 4: Rate of germination of spores of M. acridum in operational conditions of storage in different sites in Senegal (n = 5; p < 0.001) (SH: Oily suspension, SS: dry spores) 88 Diedhiou et al., Curr. Agri. Res. Jour., Vol. 2(2), 83-88 (2014) as already reported by Maniania and Una (2001). The survival rate of spores of M. acridum Fargues et al., (1997) demonstrated the existence in the 2 formulations showed a good performance of a positive correlation between virulence, spore of the dry spores within 3 months, covering the germination, mycelial growth and spore production. whole rainy season, at which crops are at risk. According to these authors, spore production In operational conditions, dry spores formulation depends of dry matter of the mycelium produced could be stored in the target sites to benefit from that is affected by climatic factors but also the culture local availability, facilitating the use of the biocontrol substrate as well. These observations seem to point in product in the field. The oil formulation in contrast, the direction of the use of fungal strains best adapted is not suited to high temperature and low moisture to the ecological conditions of the target area. This conditions for a long period. However, its use could assumption, however, should be extensively studied be justified within 1 month as the threshold for to better understand the conditions of use of M. obtaining a good efficiency is greater than 85% acridum in the areas of migration of locusts across (Junkins and Grzywacz, 2000). The position of the Africa. With a very low germination rate of spores, central storage facility in Dakar at the seaside, with poor mycelial growth and no sporulation at 37 °C, this reduced variations of temperatures while benefiting strain of M. acridum may be safe to human. These from high moisture could be the reason for the observations are consistent with the findings ofE PA prolonged good conservation of the product over (2002) and Mancebo et al., (2005) who reported no the 3 months in contrast to the inland sites. significant risk for humans and mammals, in relation with the use of M. acridum.

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